- Preparation of sphere media:
a. In serum-free and phenol red-free DMEM, add 20 ng/ml EGF, 20 ng/ml bFGF, and 1X B27 supplement. For example, to create 100 ml sphere media, add 2000 ng EGF, 2000 ng bFGF, and 2 ml of 50X B27 supplement.
b. Alternatively, add 1% FBS \(v/v) into phenol red-free DMEM in the absence of EGF, bFGF, or B27 supplement.
- Preparation of cellular subsets:
a. Aspirate media from flask containing cancer cells, wash twice in 1X PBS, and trypsinize cells.
b. Centrifuge cells at 300 x g at room temperature \(15-25°C) for 10 minutes.
c. Decant supernatant and resuspend cells carefully in sphere media prepared from Step 1.
d. Use 40µm cell strainer cap filter to obtain single-cell suspension.
e. Isolate cellular subsets of interest via FACS or MACS \(if necessary).
f. Determine cell viability via 0.4% Trypan Blue dye exclusion.
g. Seed cell suspension at 1 cell per well in ultra low-attachment 96-well plate.
As a negative control, use non-tumorigenic breast cells, such as MCF-10 or MCF-12A.
Incubate ultra low-attachment 96-well plates under standard conditions at 37°C and 5% CO2.
Using microscope (with fluorescent capability if necessary), assess wells daily for sphere formation. Allow plates to incubate for 10 days or more.
Calculate sphere-forming efficiency in the 96-well plate using the following equation:
Tumorsphere efficiency (%) = (# of spheres) / (# of wells seeded) x 100%
- Serial passaging of spheres
a. Dissociate sphere mechanically using P1000 pipette or chemically using pre-warmed trypsin-EDTA.
b. Centrifuge at 300 x g for 10 minutes at room temperature \(15°C-25°C).
c. Prepare cells in sphere media created in Step 1.
d. Seed 1 cell per well into a new ultra low-attachment 96-well plate.
e. Assess sphere-forming efficiency in secondary, tertiary, and quaternary passages using formula from Step 6.